G protein-coupled receptors (GPCRs) mediate many important physiological responses. Arrestins regulate desensitization and endocytosis of agonist-occupied GPCRs by acting as protein scaffolds for clathrin and the AP2 adaptor complex. Also, arrestin controls GPCR activation of growth signaling cascades through direct interactions with c-Src tyrosine kinase. We have recently discovered that a small region of the N-terminus of beta-arrestin2 binds to the mu2-adaptin subunit of AP2. The mu2-adaptin subunit is critical for the AP2 mediated endocytosis in clathrin-coated pits of many non-GPCR proteins that possess tyrosine phosphorylation motifs. It is the goal of this proposal to characterize using optical imaging and biochemical techniques the role of the mu-adaptin subunit and tyrosine kinases in the regulation of arrestin-mediated GPCR endocytosis. The specific aims of the proposal are: (I) To test the hypothesis that the mu2-subunit of AP2 plays a critical role in arrestin mediated GPCR endocytosis and signaling by identifying the interacting sequences and testing their importance in GPCR endocytosis and arrestin-mediated signaling. (II) To test the hypothesis that arrestin, by interacting with tyrosine kinases such as c-Src is regulated by tyrosine phosphorylation by (a) Determining the arrestin sequence where tyrosine phosphorylation can occur using arrestin derived peptides, purified arrestins and purified tyrosine kinases and assess the importance of these residues in arrestin mediated GPCR regulation This proposal will greatly our understanding of the cell physiology underlying normal health, by characterizing novel areas of arrestin biochemistry that regulate at least two major signaling pathways.